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Isoflurane induced impairment of synaptic transmission in hippocampal neurons of the guinea pig in vitro Miu, Peter 1988

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ISOFLURANE INDUCED IMPAIRMENT OF SYNAPTIC TRANSMISSION IN HIPPOCAMPAL NEURONS OF THE GUINEA PIG IN VITRO by Peter Miu B.Sc. (Hon.), The U n i v e r s i t y of B r i t i s h Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & The r a p e u t i c s ) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 © P e t e r Miu, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology & Therapeutics The University of British Columbia Vancouver, Canada Date May 9, 1988 DE-6 (2/88) MIU, P. i i A b s t r a c t T h e e f f e c t s o f a n a e s t h e t i c a p p l i c a t i o n s o f i s o f l u r a n e on HZ CA^ n e u r o n s were s t u d i e d i n i n v i t r o p r e p a r a t i o n s ( g u i n e a p i g s ) u s i n g i n t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s . V a r i o u s p a r a m e t e r s o f t h e i r e x c i t a b i l i t y s u c h as membrane e l e c t r i c a l p r o p e r t i e s , a c t i o n p o t e n t i a l s and t h e i r a f t e r h y p e r p o l a r i z i n g p o t e n t i a l s as w e l l as s y n a p t i c t r a n s m i s s i o n were d e t e r m i n e d d u r i n g b a t h p e r f u s i o n o f c l i n i c a l l y r e l e v a n t c o n c e n t r t a i o n s o f i s o f l u r a n e . C o n c e n t r a t i o n s o f i s o f l u r a n e were d e t e c t e d i n t h e b a t h w i t h 19 f l u o r i n e n u c l e a r m a g n e t i c r e s o n a n c e t e c h n i q u e s , and were f o u n d t o r a n g e b e t w e e n 0.02 and 0.3 mM. No c o n s i s t e n t e f f e c t s on t h e membrane p r o p e r t i e s w ere o b s e r v e d . When s y n a p t i c a c t i v i t y was b l o c k e d b y t e t r o d o t o x i n , i s o f l u r a n e i n d u c e d a h y p e r p o l a r i z a t i o n ( 3 - 5 mV) w i t h o u t a f f e c t i n g i n p u t c o n d u c t a n c e w h i c h was c o m p u t e d f r o m t h e v o l t a g e r e s p o n s e s t o i n j e c t i o n s o f h y p e r p o l a r i z i n g c u r r e n t p u l s e s and t h e s l o p e s o f c u r r e n t - v o l t a g e r e l a t i o n s f o r e a c h c e l l . R e s p o n s e s t o d e p o l a r i z i n g p u l s e s r e v e a l e d t h a t t h e t h r e s h o l d , a m p l i t u d e and d u r a t i o n o f t h e e v o k e d s p i k e s w e r e n o t g r e a t l y a l t e r e d , a l t h o u g h r e p e t i t i v e s p i k e f i r i n g was s u p p r e s s e d i n a d o s e - d e p e n d e n t manner by i s o f l u r a n e . S i m i l a r l y , t h e a m p l i t u d e and d u r a t i o n o f t h e l o n g - l a s t i n g h y p e r p o l a r i z a t i o n s f o l l o w i n g t h e e l i c i t a t i o n o f m u l t i p l e (4 o r 5) s p i k e s w e r e r e d u c e d i n a r e v e r s i b l e a n d d o s e - d e p e n d e n t manner. R e d u c t i o n s i n a m p l i t u d e and d u r a t i o n o f e x c i t a t o r y a n d i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l s e v o k e d b y e l e c t r i c a l s t i m u l a t i o n o f s t r a t u m r a d i a t u m w ere o b s e r v e d ; t h e s e e f f e c t s a l s o were s t r i c t l y d e p e n d e n t on t h e d o s e , as w e l l as on d u r a t i o n o f t h e a p p l i c a t i o n . T h e s e i n v e s t i g a t i o n s h a v e r e v e a l e d t h a t i s o f l u r a n e i n t e r f e r e s w i t h s y n a p t i c t r a n s m i s s i o n i n t h e h i p p o c a m p a l s l i c e p r e p a r a t i o n and s u g g e s t t h a t p r e s y n a p t i c a c t i o n s on t r a n s m i t t e r r e l e a s e , i n a d d i t i o n t o p o s t s y n a p t i c e f f e c t s c o n t r i b u t e t o t h e m e c h a n i s m s o f t h e a n a e s t h e t i c s t a t e i n d u c e d b y i s o f l u r a n e . MIU, P. i l l TABLE OF CONTENTS CHAPTER Page 1 INTRODUCTION 1 1.1 Historical background 1 1.2 Physiological definit ion of consciousness 1 1.3 Anatomical correlates of consciousness 2 1.3.1 Reticular activating system 2 1.3.2 Cerebral hemispheres 3 1.4 Conscious experience 3 1.4.1 Mental processes 4 1.4.2 Molecular mechanism 4 1.5 Objectives 5 2 METHODS 7 2.1 Animal preparation 7 2.2 Administration of isoflurane 9 2.3 Determinations of isoflurane concentrations 9 2.4 Recording techniques and arrangement 10 2.5 Data acquisition 11 2.6 Computer analysis 13 2.7 Stat ist ics 13 3 RESULTS 14 3.1 Electr ical membrane properties of CAi pyramidal ce l l s 14 3.2 Isoflurane concentrations 14 3.3 Spikes and membrane potentials 18 3.4 Input resistances and voltage-current relations 20 3.5 Postspike afterhyperpolarizing potentials (AHPs) 28 3.6 Synaptic transients 35 MIU, P. i v CHAPTER Page 4. DISCUSSION 44 4.1 I s o f l u r a n e concentrations 44 4.2 Postsynaptic m o d i f i c a t i o n s 4b 4.2.1 Resting p o t e n t i a l s and membrane conductances 45 4.2.2 A f t e r h y p e r p o l a r i z i n g p o t e n t i a l s 47 4.3 Presynaptic m o d i f i c a t i o n s 49 4.3.1 Axonal conduction 49 4.3.2 Synaptic transmission 50 4.4 Mechanism of anaesthesia 51 4.4.1 Membrane e f f e c t s 51 4.4.2 Transmitter r e l e a s e 52 5 SUMMARY AND CONCLUSIONS 55 6 REFERENCES 57 LIST OF FIGURES MIU, P. v Page 1. Schematic drawing o f a s i m i l a r t y p e o f p e r f u s i o n chamber f o r i n t r a c e l l u l a r r e c o r d i n g s . 8 2. Schematic drawing o f t h e hippocampal s l i c e . 12 3 . I s o f l u r a n e c o n c e n t r a t i o n s as determined by ^ f l u o r i n e n u c l e a r magnetic r e s o n a n c e t e c h n i q u e s (l^F-NMR). 16 4. I s o f l u r a n e a c t i o n s on p a s s i v e e l e c t r i c a l membrane p r o p e r t i e s o f hippocampal CA^ p y r a m i d a l c e l l s . 21 5. V o l t a g e - c u r r e n t (V/I) r e l a t i o n s h i p s o f CA^ p y r a m i d a l c e l l s . 24 6. V o l t a g e - c u r r e n t (V/I) r e l a t i o n o f one C A i py r a m i d a l c e l l , i n t h e p r e s e n c e o f 1 yM TTX. 2b 7 . P o s t s p i k e a f t e r h y p e r p o l a r i z a t i o n s (AHPs). 29 8. E f f e c t s o f i s o f l u r a n e on AHPs o f 18 CA^ p y r a m i d a l neurons. 33 9. I s o f l u r a n e r e d u c e s t h e a m p l i t u d e s o f EPSPs i n a dose- and time-dependent manner. 37 10. Time c o u r s e o f i s o f l u r a n e a c t i o n and r e c o v e r y on evoked EPSPs. 39 11. E f f e c t s o f i s o f l u r a n e on i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l s ( I P S P s ) . 42 MIU, P. v i LIST OF TABLES Page 1. Number o f i s o f l u r a n e a p p l i c a t i o n s . 15 2. E f f e c t s o f i s o f l u r a n e on spontaneous f i r i n g o f CA^ p y r a m i d a l neurons. 19 3. E f f e c t s of i s o f l u r a n e on the a m p l i t u d e s o f AHPs o f CAj_ p y r a m i d a l neurons. 31 4. E f f e c t s o f i s o f l u r a n e on t h e d u r a t i o n s o f AHPs o f CA^ p y r a m i d a l neurons. 32 5. E f f e c t s o f i s o f l u r a n e on t h e e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s of CA]_ p y r a m i d a l neurons. 36 MIU, P. v i i ACKNOWLEDGEMENTS I am grateful to Dr. E. Puil for allowing me tne opportunity to study under his guidance, and his patience and encouragement throughout my degree programme. I like to thank Boris for his help in the computer portion of my work. I wish him best of luck in his future endeavor. Special thanks to Igor and Samuel for their humor which made my lab work bearable. Last but not least, members and staff of the Department of Pharmacology & Therapeutics for being so nice to me. Also, thanks to the Department of Chemistry for helping me out with the 19 fluorine nuclear magnetic resonance analysis. I LIKE TO DEDICATE THIS THESIS TO MY MOM FOR HER LOVE, PATIENCE, AND SUPPORT THROUGHOUT MY POSTSECONDARY EDUCATION AND LAST BUT NOT LEAST MY BEST FRIEND MAHSA FOR BELIEVING IN ME (SDIMBYAH) MIU, P. 1 1. INTRODUCTION 1.1 Historical background In the early 1800s, surgical procedures were performed only with great risk to the patient. Relief from pain during such operations was attempted by administering alcohol, narcotics such as hashish, and opiates. In desperation, the patient was either rendered unconscious by a blow to the head, or was restrained by force (for those who were less fortunate). Although nitrous oxide (laughing gas) was available, i ts use was 'purely' for entertainment purposes (Goodman and Gilman, 1985; Mi l le r , 1986) The analgesic property of nitrous oxide was discovered in 1844 by a dentist, Horace Wells. During an exhibition of i ts exhilarating properties, he observed that a participant accidently injured himself without feeling any pain while under the influence of nitrous oxide. This discovery enabled Wells to perform painless tooth extraction, using nitrous oxide as the sole analgesic agent. Unfortunately, attempts to persuade his colleagues about these therapeutic effects were unsuccessful. William T.G. Morton in 1846 introduced diethyl ether as the hypnotic agent for surgical procedures, which subsequently became the agent of choice for general anaesthesia. Nitrous oxide later received extensive use in dental and surgical practices in the United States. Since that time, there have been many developments of new and much more specif ic anaesthetic/analgesic substances (Goodman and Gilman, 1985). 1.2 Physiological definition of consciousness A fascinating feature of the general anaesthetic c lass i f icat ion is the lack of a stringent requirement in the chemical structure of an agent and i ts anaesthetic act iv i ty (SAR). However, the ultimate effect induced by such agents is unconsciousness, i . e . , the anaesthetic state which is c l i n i -cal ly defined as a collection of anaesthetic effects in humans. MIU, P. 2 Presently, an exact definition of consciousness is elusive because of the discrepencies in interpretations of consciousness by proponents of various d isc ip l ines, e . g . , philosophy, psychology, and physiology. Human consciousness can be divided into two dist inct components, the level of consciousness (or wakefulness), and awareness. An index of the level of consciousness is alertness which is maintained by the reticular activating system and i t s connections to the cerebral cortex. Indices for awareness include attention, expectations, learning, and memory. These attributes of awareness are inextricably l inked, and dependent on, anatomically defined regions of the brain such as the limbic system and other specialized regions of the cerebral hemispheres. Hence, communication between various brain structures (e .g . , the cerebral cortex and the hippocampus) is crucial for mental processes which invariably include memory. Therefore, a generalized suppression of neuronal exc i tabi l i ty in the neocortex and reticular activat-ing system, induced by lesioning or drugs, may produce alterations in cons-ciousness (Ropper and Martin, 1983). 1.3 Anatomical correlates of consciousness 1.3.1 Reticular activating system. The level of wakefulness is main-tained by the reticular activating system, consisting of nuclei located in the medial tegmental gray matter of the brainstem which extend from the medulla to the posterior diencephalon. Early experiments demonstrated that stimulation of the reticular formation desynchronizes the electr ical activ-ity (EEG), consistent with awakening from sleep (Moruzzi and Magoun, 1949). In contrast, lesions of the mesencephalon or diencephalon abolish the EEG activation associated with wakefulness (Lindsley et a l , 1949; Lindsley et a l , 1950). Subsequent experiments have revealed that cortical arousal is regulated partly by diffuse and nonspecific ascending projections from the reticular formation direct ly , or indirectly through the intra!aminar and MIU, P. 3 related thalamic nuclei (Starzl et a l , 1951a,b). Mence, the attenuation of wakefulness by hypnotic agents has been proposed to be produced by a depression of the reticular activating system (Lindsley et al_, 1949; Lindsley et a l , 1950; French et a l , 1953; Arduini and Arduini, 1954; Mori et al_, 1972; Shimoji et a l , 1984). 1.3.2 Cerebral hemispheres. Despite the powerful influence of the reticular formation, wakefulness also depends primarily on the overall functional state of the cortex. For example, an ischemic condition of the brain induces an uncoupling between neuronal metabolism and electr ical act iv ity thereby resulting in the disappearance of useful cerebral function or coma (Krnjevic, 1975). With the development of radioactive glucose detection techniques, various investigators have demonstrated that anaes-thesia is induced by an 'uniform' depression of brain metabolic act iv i ty ; nevertheless, some regions of the brain respond differently in their changes of metabolism (Sokoloff et a l , 1977; Savaki et a l , 1983; Peschanski et a l , 1986). Functional paralysis of cerebral structures by anaesthetics begins with the cortex and descends to basal ganglia, cerebellum, spinal cord, and medulla (Vickers et a l , 1984). Other factors which contribute to cortical dysfunction resulting in alterations in wakefulness include lesion, epilep-sy, and hypoglycemia. 1.4 Conscious experience Physical insults such as diseases, and trauma to the brain commonly lead to an permanent, i . e . , i r revers ib le , loss of mental awareness of the internal and external milieux, and subsequent attenuation of cognitive func-tions. Anaesthetic actions are reversible and furthermore, awareness during surgery (Utting, 1982) as well as the revers ib i l i ty of anaesthesia indicate a less extensive, and possible more selective, assault on cortical functions in contrast to that observed with physical damage. The transient postanaes-MIU, P. 4 thetic depression of cognitive functions (e .g . , learning and memory) is a re lat ively common observation in patients; however, prolonged inhibition of cerebration ( i . e . , the residual depression of the central nervous system --CNS), lasts several hours, and is more prevalent or pronounced in elderly patients (Davenport, 1986). Hence, the effects of anaesthetic state on hum-an cognitive functions may terminate less rapidly than other, more obvious c r i ter ia for consciousness, e . g . , the level of wakefulness (Miu and P u i l , 1988). 1.4.1 Mental processes. Learning and memory are a consequence of asso-ciations between sensory experiences and diverse brain structures. The exact regions of the brain in which these processes take place are s t i l l a mystery. Nevertheless, learning is assumed to be associated with cerebral hemispheres, despite the observations that in some animal species without cortex, the capability of learning is retained. In humans, impairment in the capacity for new learning is associated with the lesioning or destruc-tion of primarily the neocortex. Furthermore, c l in ica l studies have pro-vided valuable information on the relationship between the location of cortical lesions and the resulting cognitive deficiency. For example, Meyer and Yates (1955) noted evidence of defective learning ab i l i ty with temporal lobe epilepsy. Later, Scovil le and Milner (1957) demonstrated that the temporal lobes and hippocampal formations are involved in learning and retrieval of learned responses from memory. 1.4.2 Molecular mechanism. The molecular mechanism of learning and memory has been the subject of intensive examinations, and the results ind i -cate that learning or conscious experience produces anatomical, biochemical, and electrophysiological changes at synapses. Eccles (1965) hypothesized that conscious experience fac i l i tates growth of pre-existing synapses. This hypothesis was supported by the findings that rats placed in an enriched Mill, P. 5 environment ( i . e . , presence of stimulating objects) develop a greater of the cerebral cortex, i . e . , in actual cortical weight (Rosenzwieg and Bennett, 1969) as well as a greater numbers of spines per unit of length of dendrite (Globus et a l , 1973) than animals placed in an impoverished environment. In addition, other investigations have shown that s ignif icantly greater dendri-t i c branching develops in animals exposed to the enriched environment (Volkmar and Greenough, 1972; Greenough and Volkmar, 1973). The transforma-tion from temporary, to more permanent synaptic contacts occurs with repeat-ed activation of the same pathways, hence the concept of consolidation of memory. Cotman and Nieto-Sampedro (1982) concluded that synapses throughout the nervous system have a continuously high turn over rate, and that learn-ing stimulates this ongoing p last i c i ty . Behavioral p last ic i ty and cognitive processes, manifest as electr ical signals, occur in the associative cortex, different from the primary motor and sensory area (Pandya and Seltzer, 1982). Also, the extensive inter-con-nections between the associative cortex and hippocampus (Teyler and DiScenna, 1984b) suggest that the learned responses are transmitted to the hippocampus where consolidation occurs by a mechanism of long-term potentiation (LTP) (Teyler and DiScenna, 1984a). 1.5 Objectives Pauling (1961) believed that both consciousness and memory were the result of electr ical osci l lat ions in the brain. This view is extended by the proposal that self-consciousness (awareness of internal self) would not occur in the absence of memory (Krnjevic, 1974; Popper and Eccles, 1977). Because of the l i ke ly poss ib i l i t ies that anaesthetics affect the consolida-tion and retrieval of learned responses and that their persistent effects produce the slow return of cognitive processes during the postoperative period, we hypothesized that anaesthetic agents may disrupt mnemonic commun-ication by direct actions on neocortical and archicortical structures. MIU, P. 6 Although the importance of the neocortex in these processes appears paramount (cf. above), the in vitro slice preparation of the hippocampal formations in experimental animals confer many advantages over similar preparations of neocortex. For example, the well-defined anatomical organ-ization of the hippocampus allows easy visualization and identification of the perikarya for experimental studies involving intracellular penetration with microelectrodes. The lamellar organization of fibres in the hippo-campus fac i l i t a t e s the isolation of selective pathways that can be stimu-lated, i.e., stimulation in the stratum radiatum evokes orthodromic responses in the CAj c e l l s . An important feature of the hippocampus, or at least the slice preparations of hippocampus, is the relative resilience to hypoxic conditions inadvertly incurred by excision of the tissue. The choice of anaesthetic in these investigations was isoflurane which is a relatively new agent that has come into prominent cl i n i c a l use during the past 5 years in North America. Hence, the effects of isoflurane were investigated on the membrane electrical properties and activities of pyrami-dal cells in in vitro slice preparations of the hippocampus. The emphasis in these investigations was to determine, using intracellular recording techniques, the actions of isoflurane on the membrane excitabilities and synaptically evoked potentials in the CA^ neurons. Specifically, we were interested in determining the actions of the anaesthetic on excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). Furthermore, 19 fluorine-NMR techniques were used to estimate the exact concentrations of isoflurane in the solution bathing the hippocampal tissue. MIU, P. 7 2. METHODS 2.1 Animal preparation The experiments were performed on in vitro slice preparations of hippo-campus obtained from albino guinea pigs of either sex, weighing 200-300 g. An animal was anaesthetized i n i t i a l l y with 4% halothane in a closed 2-liter chamber. Following loss of the righting reflex, 4 % (v/v) halothane was administered by mask (O2 flow rate, 1 liter/min). Tracheotomy was per-formed in order to assist ventilation, and halothane was subsequently delivered endotracheally. Sometimes, a r t i f i c i a l ventilation was employed. The concentration of halothane required for the maintanence of anaesthesia was titrated against spontaneous ventilation and responses of the animal to painful stimuli induced by surgical manipulations. The usual dose of halo-thane administered at this stage varied from 1 to 2% (v/v). The animal then was placed in a stereotaxic head holder and an extensive bilateral cranio-tomy was performed. Care was taken during this procedure so as to preserve the integrity of the dura since i t was consistently observed that tearing of the dura while removing the skull always led to uncontrollable subdural hematoma. After removal of the dura, the cerebral cortices were aspirated to reveal the underlying hippocampi. Each hippocampus was undercut in one motion with a large scalpel blade and quickly immersed in pre-cooled K4°C) a r t i f i c i a l cerebrospinal fluid (ACSF) oxygenated with 95/5 % gaseous mixture of 0 2/C0 2. The constitu-ents of the ACSF were (in mM): NaCl, 124; KC1, 3.75; KH2P04, 1.25; MgS04.7H20, 2; CaCl .2H20, 2; dextrose, 10; NaHC03, 26. Thick (400 Lim) transverse slices of the hippocampus were cut with a Mcllwain t i s -sue chopper. One slice was 'sandwiched' in nylon mesh and placed in the recording chamber (Fig. 1). The remaining slices were kept at room temper-MIU, P. 8 Aspi ro lor FIG. 1. Schematic drawing of a s i m i l a r type of p e r f u s i o n chamber f o r i n t r a c e l l u l a r r ecordings. MIU, P. 9 ature (22-24°C) in ACSF, bubbled continuously with 95/5% of 0 2/C0 2 mix-ture, until needed. The temperature of the bath was maintained at 32°C by a thermoregulator throughout the experiment. The slice was perfused in the recording chamber at a constant rate of 2-3 ml/min with oxygenated ACSF (pH 7.4). Humidified gas (95% Oy 5 % C02) was passed into the atmos-phere immediately above the sli c e . 2.2 Administration of isoflurane Since the total volume of the recording chamber was 1 ml, the perfusate was rapidly exchanged within about 1 min, with fresh ACSF solution. Iso-flurane was applied in the concentrations indicated by the output settings of several Fluotec-3 vaporizers using 95% C^, 5% CIV, at a flow rate of more than 500 ml/min. The ACSF in several reservoirs consisting of inverted syringes (60 ml volume) was bubbled with the gas mixture for a minimum of 20 min to ensure a maximal equilibration with the anaesthetic at a designated concentration before its application by perfusion to the s l i c e . 2.3 Determinations of isoflurane concentrations The concentrations of isofurane in the bathing solution were determined 19 19 using the fluorine-nuclear magnetic resonance ( F-NMR) technique. Aliquots (1.6 ml) of the perfusate, pre-equil ibrated with a particular con-centration of isoflurane indicated by the setting of the calibrated vaporiz-er output, were collected from the recording chamber with an air-tight syringe and injected into MMR sampling tubes containing 0.3 ml of 20 mM t r i -fluroacetic acid (TFA) (final concentration of TFA in the sampling tube was 2.5 mM) and 0.5 ml of deuterium oxide. The MMR tubes were then tightly capped and analyzed in the Department of Chemistry at room temperature using an MMR spectrometer (270 MHz). Field lock was used with D20 as the lock-ing signal. Signals were averaged from 3000 scans/sample with the sweep width set to 6000 Hz Sometimes, more signals were averaged to reduce back-MIU, P. 10 ground noise. Proton-decoupling was used with an offset frequency of 4000 19 Hz. The F-NMR signals generated from TFA (internal standard) and iso-flurane were Fourier transformed, and the size of individual peak integrated. The concentration of isoflurane in the perfusate was determined by taking the ratio of the integrals from the internal standard and the second peak, -4.8 ppm, (i.e. tri-fluorine atoms of isoflurane), multiplied by the con-centration of the internal standard: [IFL] = IFL/TFA x [TFA]. 2.4 Recording techniques and arrangement Intracellular recordings began approximately 1 hr after placing the slice in the recording chamber. Microelectrode, mounted on a Narishige micromanipulator, was positioned into the CA^  region of the hippocampal slice under visual guidance with a dissecting microscope. The microelec-trode was rapidly advanced into the f i r s t 150 um of the slice because i t had been assumed that most of the pyramidal cells near the surface were either damaged or destroyed by the tissue chopper. Thereafter, the microelectrode was lowered with smaller advancing increments (approximately 1 um steps) until a cell was found. Successful penetration of the recording electrode was achieved by 'buzzing' the cell with the electrode capacitance neutraliz-ation control on the amplifier. Conventional intracellular recording techniques were used with a WPI amplifier (Model M701) connected to an oscilloscope (Tektronic RM 565). CA^ neurons were impaled with single barrel glass micropipettes (1 mm diameter tubing) containing 3 M KC1 or with 4 M potassium acetate (KAc) for experiments on evoked synaptic potentials. The tip resistances of 3 M KC1 electrodes, measured in the tissue before penetrating the c e l l , ranged from 40 to 60 Mfi, whereas 4 M KAc electrodes ranged from 60 to 80 Mfi. Membrane input resistances were estimated with bridge-balance techniques from voltage MIU, P. 11 responses to intracellular injections of constant current pulses (100 ms duration delivered at 1 Hz). Resting membrane potential was monitored con-tinuously with a chart recorder, and zero voltage level was determined after withdrawing the electrode from the c e l l . Amplified intracellular signals were digitized (PCM-1 digital VCR-in-strumentation recorder adaptor) and stored on super VHS tape with a Canon recorder (model VR-HF 600) for off-line playback and analysis by computer. Some of the recorded intracellular signals (in analog form) were analyzed with a waveform recorder (Biomation 805) which has the capability of digi-tizing the input voltage sweep into 2048 points thus permitting manual con-trol of digital-to-analog conversion for display on the oscilloscope, or reproduction of the sweep on paper of an X-Y pen recorder (Hewlett-Packard 7015B). For experiments on synaptic potentials, a tungsten bipolar stimulating electrode was place in the stratum radiatum region of the hippocampal slice (Fig. 2). Stimulation frequency was set to 0.2 Hz. and the stimulation strength ranged from 10-15 V. 2.5 Data acquisition Intracellular voltage signals, played back from Canon tape recorder, were fed into 'ERAT', an interface panel, which converts the input signals in the analog form to the digital form. The data were stored temporarily in the memory bank of MINC-23 computer during the data acquisition. After com-pletion of this phase, the f i l e s were transferred and stored "for the actual analysis procedures in another computer (PDP 11/44) which has a larger memory capacity. ALV: Alveus COM: Commissural fibres FIM: Fimbria FIS: Hippocampal fissure GC: Granule cell body layer MF: Mossy fibres PP: Perforant path SCH: Schaffer collaterals FIG. 2. Schematic drawing o f t h e hippocampal s l i c e . MIU, P. 13 2.6 Computer analysis Individual traces of synaptic transients in the raw data f i l e of MINC were selected, and approximately 17 to 25 traces in each of the three groups (control, isoflurane, and recovery) were chosen from the raw data f i l e for the averaging program. A new f i l e was subsquently created for the averaged traces, and various parameters of the synaptic transients from these aver-aged traces were analyzed, e.g., amplitude of both EPSPs and IPSPs, and changes in resting potentials. 2.7 Statistics Most data were tested s t a t i s t i c a l l y with one-way analysis of variance (ANOVA) for overall significance. Group comparisons were performed using post hoc comparison test. Sample size of all the experiments was 5 or more, unless otherwise stated. The accepted level of significance is p<0.05. MIU, P. 14 3. RESULTS 3.1 Electrical membrane properties of CA^ pyramidal cells Only cells with stable resting potentials and a relative constant input resistance measured over 20 min were selected for isoflurane administration. Stable recordings were obtained from 82 out of 100 CA^ pyramidal c e l l s . Within 5 to 10 min following cell penetration by the recording electrode, resting potential usually hyperpolarized and eventually stabilized at a more negative value. Concomitantly, input resistance, measured by injecting 100 ms hyperpolarizing pulses, also increased and thereby indicated proper seal-ing of the membrane around the microelectrode. The mean resting potential obtained from a random sampling of 23 cells was -65.4 ± 1.3 mV. Input resistance was usually greater than 50 Mfl (cf. Fig. 4). Both spontaneous, and single spikes evoked by intracellular injections of depolarizing cur-rent, had peak amplitudes of more than 80 mV. The recordings generally lasted 1-5 hours, and most cells were exposed to more than one dose of iso-flurane (Table 1). 3.2 Isoflurane concentrations To ascertain the amount of isoflurane that had been dissolved in the perfusate, samples of ACSF containing different concentrations of isoflurane were collected from the recording chamber and analyzed by spectrometer with 19 radiofrequency of 270 MHz. An example of the F-NMR spectrum obtained from 4% isoflurane dissolved in ACSF is illustrated in Fig. 3A. The f i r s t peak represents the internal standard (2.5 mM TFA). The second and third peaks, downfield from the TFA peak, occurring at -4.8 ppm and -10.8 ppm respectively, represent t r i - and di-fluorine atoms of isoflurane. The concentration of isoflurane in the perfusate was calculated by taking the IFL cone. No. of No. of (Vol. %} c e l l s applications 0.5 15 28 1.0 18 28 1.5 5 6 2.0 20 49 3.0 12 20 4.0 12 17 Total 82 148 Table 1 . Number of i s o f l u r a n e a p p l i c a t i o n s . MIU, P. FIG. 3. I s o f l u r a n e c o n c e n t r a t i o n s as determined by 19 . . 1 9 f l u o r i n e n u c l e a r magnetic resonance techniques ( F-NMR). 19 . . . (A) F-NMR s p e c t r a of ACSF c o n t a i n i n g 4% i s o f l u r a n e which was c o l l e c t e d from r e c o r d i n g chamber. The f i r s t peak (0 ppm) re p r e s e n t s TFA, i n t e r n a l standard. The second (-4.8 ppm) and t h i r d (-10.8 ppm) peaks r e p r e s e n t t r i - and d i - f l u o r i n e atoms of i s o f l u r a n e r e s p e c t i v e l y . S p e c t r a analyzed by spectrometer with r a d i o f r e q u e n c y 270 MHz. (B) T h e o r e t i c a l and experimental c o n c e n t r a t i o n s of i s o f l u r a n e p l o t t e d a g a i n s t v a p o r i z e r output s e t t i n g s . MIU, P. 17 MIU, P. 18 ratio of the integrals under the peaks (cf. Methods), and the vaporizer output settings from 0.5-4% were subsequently converted to give correspond-ing NMR concentrations which ranged from 0.02 to 0.3 mM (Fig. 3B). Also included in Fig. 3B is the calculated or theoretical values. Comparison of the theoretical values with those empirically determined reveals that 40 % of isoflurane was lost in the perfusate. This can be attributed to d i f f i c u l t i e s in the collection procedure, e.g. improper seal-ing of the sampling tube, leakage before actual measurement, etc. Also, the large concentration difference between the solution containing isoflurane and the atmosphere above the orifice of the recording chamber may f a c i l i t a t e rapid evaporation. This process i s , of course, facilitated by the large concentration gradient existing between the atmosphere and the perfusate containing dissolved isoflurane. In addition, the l i p i d solubility of the slice preparation is greater than that of the ACSF. 3.3 Spikes and membrane potentials Spontaneous fi r i n g in 20 pyramidal c e l l s , exposed to approximately 6 min of various concentrations of isoflurane, were analyzed, and the results are summarized in Table 2. Isoflurane concentrations ranging from 0.5 to 4% were applied. At low doses, three different responses were observed, i.e., reduction, no change, and augmentation in the rate of spontaneous f i r i n g . The percentage of cells that responded to 0.5 % isoflurane applications with a reduction in the rate of spontaneous f i r i n g was 33 % (Table 2). With increasing isoflurane concentrations, however, the number of cells with a reduced f i r i n g rate was greater and the reduction reached a maximum value of 60% in the presence of 3 % i s o f l urane. In addition, the number of cells with unaltered f i r i n g rates was decreased with increasing anaesthetic concentra-tions. That i s , with applications of 3 and 4%, isoflurane either enhanced or decreased the rate of spontaneous f i r i n g . The amplitudes and durations MIU, P. IFL cone. C e l l no. Spontaneous No. a p p l i c a t i o n s (Vol. %) f i r i n g r e s u l t e d in reduced f i r i n g (%) 0.5 1 4 2 • 3 + 2 0 3 0 33 1.0 4 6 1 5 1 40 2.0 3 -10 -11 -12 -13 -14 -7 + 7 + 7 0 8 0 9 0 3.0 6 12 17 15 16 + + 60 4.0 6 13 20 18 19 60 -: reduction +: augmentation 0: no change T a b l e 2 . E f f e c t s o f i s o f l u r a n e on spon taneous f i r i n g o f CA p y r a m i d a l n e u r o n s . MIU, P. 20 of single spikes were unaltered by isoflurane administration (data not shown). Various concentrations of isoflurane were applied to 50 pyramidal c e l l s . The changes in their resting potentials measured at 6 min of isoflurane perfusion are summarized in Fig. 4A. The columns in this figure represent the percentage of the total number of applications for the particular appli-cation. Isoflurane administration in low concentrations did not e l i c i t a change in membrane potential in many neurons. However, the majority of neurons were depolarized by low doses, whereas doses of 2% and higher hyper-polarized most cell s . The only exception was the 1% applications where a majority of the cells responded to isoflurane with no change in membrane potential. In several experiments, 1 nM tetrodotoxin (TTX) was added to the perfusate to eliminate the influences of released synaptic transmitter on the resting potential. The results showed that 12 out of 12 cells were hyperpolarized by isoflurane in concentrations of 1.0-4.0% (data not shown). One cell responded to 4%isoflurane application with membrane hyperpolariza-tion while the other cell depolarized. 3.4 Input resistances and voltage-current relations Instantaneous membrane input resistance was measured by injecting hyperpolarizing current pulses at 1 Hz through a bridge balanced recording electrode into the soma of CAj pyramidal c e l l s . Subsequent voltage deflection was measured, and membrane input resistance was calculated using the Ohm's law: V = IR where V is membrane voltage response to current injection, I is the current injected, and R is the input resistance. Isoflurane, in concentrations ranging from 1.0 to 3.0%, did not reduce input resistance significantly (Student t-test). However, input resistance MIU, P. FIG. 4. I s o f l u r a n e a c t i o n s on p a s s i v e e l e c t r i c a l membrane p r o p e r t i e s of hippocampal CA^ pyramidal c e l l s . (A) D i s t r i b u t i o n of pool e d r e s t i n g p o t e n t i a l responses (82 neurons) to v a r i o u s doses of i s o f l u r a n e . R e s t i n g p o t e n t i a l s recorded a f t e r 6 min of i s o f l u r a n e p e r f u s i o n . KC1 e l e c t r o d e . (B) Input r e s i s t a n c e v a l u e s o f CA 1 c e l l s , obtained from s m a l l (<10 mV) v o l t a g e responses t o h y p e r p o l a r i z i n g c u r r e n t p u l s e s . Means ± SEM. MIU, P. 22 tfl c o IC <0 o 60-- 40-ft a Q 20-Hyperpolarization No change I—I Depolarization s s K N \ N s r 0.5 1.0 1.5 2.0 3.0 4.0 B 150.0T (ZD Control 6 min KNN 6 min recovery o C 3 ft C 100.0--I 50.0-• 0.0-1 A rl 1 j 0.0 1.0 2.0 3.0 4.0 Isoflurane concentration (Volume %) MIU, P. 23 was reduced significantly with the 4% dose. It is conceivable that the observed reduction in input resistance may not be a direct consequence of anaesthetic action since large spontaneous depolarization of unknown origin occurred in 2 out of 7 cells studied, thereby contributing to a large decrease (i.e., rectification) in the mean input resistance. These data are summarized for 19 cells in Fig. 4B. In addition to the instantaneous input resistance measurements, voltage-current relations were also examined in the presence of isoflurane. A total of 7 cells were exposed to 12 min applications of 2% isoflurane by perfusion. Membrane voltage deflections corresponding to increasing hyper-polarizing current pulses were measured and calculated using the Ohm's law decribed earlier. The data were pooled and summarized in Fig. 5A which shows that the slope resistances were not altered significantly. The magni-tude of the reduction in slope resistance was approximately the same as that measured by the steady-state voltage response to constant current injection (cf. Fig. 4B). The voltage value at the point where the control slope intercepts with the slope obtained during anaesthesia is 20 mV, which when added to the average resting potential of these cells gives -75.5 mV. Since the theore-tical reversal potential for potassium is approximately -80 mV, the close approximation of the empirical value suggests that perhaps isoflurane may alter resting K+-conductance (g K). The vol tage-current relations were also studied in the presence of 1 uM tetrodotoxin (TTX). Isoflurane was applied 4 times to 2 c e l l s , and the results are shown in Fig. 5B. In con-trast to the previous voltage-current relations, there is slight membrane rectification with large hyperpol arizing current injections. Nonetheless, a significant alteration in slope resistance also was not observed with TTX-blockade of endogenous synaptic a c t i v i t i e s . In Fig. 6, 6 min of 3% MIU, P. 24 FIG. 5. V o l t a g e - c u r r e n t (V/I) r e l a t i o n s h i p s of CA 1 pyramidal c e l l s . Open c i r c l e s r e p r e s e n t the peak v o l t a g e responses i n ACSF, and c l o s e d c i r c l e s r e p r e s e n t the peak voltage responses recorded a f t e r 12 min of 2% i s o f l u r a n e p e r f u s i o n . (A) Steady-state v o l t a g e responses t o h y p e r p o l a r i z i n g c u r r e n t i n j e c t i o n s (7 neurons) i n the absence of TTX (1 i UM) . (B) S t e a d y - s t a t e v o l t a g e responses t o h y p e r p o l a r i z i n g c u r r e n t i n j e c t i o n s (4 neurons) i n the presence of TTX (1 VM). Means (2 t o 5 c e l l s each) ± SEM are given i n neurons t h a t were i n j e c t e d with i d e n t i c a l c u r r e n t p u l s e s . MIU, P. 25 MIU, P. 26 FIG. 6. V o l t a g e - c u r r e n t (V/I) r e l a t i o n of one CA^ pyramidal c e l l , i n the presence of 1 UM TTX. Open c i r c l e s represent the peak v o l t a g e responses i n c o n t r o l ACSF, and c l o s e d c i r c l e s r e p r e s e n t the peak v o l t a g e responses re c o r d e d a f t e r 6 min of 3% i s o f l u r a n e p e r f u s i o n . MIU, P. 28 isoflurane perfusion, again, did not alter membrane slope resistance significantly. With injections of depolarizing current pulses, a slight outward rectification was observed. This figure further illustrates that this cell did not show anomalous rectification in either the depolarizing or hyperpolarizing direction, when compared to the two previous cells in which 2% isoflurane had been administered. Hence, the electrical properties of the membrane were assumed to obey Ohm's law. 3.5 Postspike afterhyperpolarizing potentials (AHPs) AHPs were generated by injecting suprathreshold depolarizing current pulses once every 10 s to e l i c i t multiple spikes, i.e. usually 4 or 5 spikes. The spikes in each train were usually more than 80 mV amplitude. The average peak amplitude of the AHPs in ACSF was 3.1 ± 0.2 mV (n=35) (cf. Table 3), and the average duration of the AHPs was 4.1 ± 0.4 mV (n=28) (cf. Table 4). Both the amplitudes and durations of the AHPs were measured in 20 cells exposed to multiple applications of various concentrations of isoflur-ane. In the representative cell of Fig. 7, the amplitudes of the AHPs were reduced at 6 min of 2% isoflurane perfusion. Full recovery to control val-ues was observed in this c e l l , and 17 other neurons at 6 min after returning the perfusion to normal ACSF. A summary for these cells is given in Fig. 8A, and Table 3 (Tables 3 and 4 represent the actual values of AHP amplitude and duration, in mV and seconds). As indicated in the Figure, the peak amplitude was slightly reduced at 3 min of isoflurane perfusion. However, profound reductions in the peak amplitude of the AHPs were observed at 6 min of isoflurane perfusion. The magnitude of reduction, obtained with applica-tion of 2-4 % isoflurane, was dose-dependent and s t a t i s t i c a l l y significant (Student t-test). In contrast, dose-dependent reductions in AHP durations were not observed with increasing isoflurane concentrations (Fig. 8B, and Table 4). •MIU, P. FIG. 7. P o s t s p i k e a f t e r h y p e r p o l a r i z a t i o n s (AHPs). AHPs were e l i c i t e d by p a s s i n g d e p o l a r i z i n g c u r r e n t p u l s e s (0.1 Hz) of 4 nA, 100 ms i n a CA^ neuron. 6 min p e r f u s i o n of 2% i s o f l u r a n e reduced both the amplitudes and d u r a t i o n s of AHPs. F u l l r e c o v e r y t o c o n t r o l values was observed at ~6 min a f t e r r e t u r n i n g the p e r f u s i o n t o normal ACSF. KC1 e l e c t r o d e r e c o r d i n g . MIU, P. 30 C o n t r o l 2% i so f lu rane Recovery 5 mV 0.9 nA 5 s MIU, P. 31 AHP a m p l i t u d e (mV ± SEM, no. o f a p p l i c a t i o n s ) IFL P e r f u s i o n t i m e (min) ( V o l . C o n t r o l I s o f l u r a n e R e c o v e r y %) 3 6 6 0.5 3.0 ±0.6 (5) 2.9 ±0.7 (5) 2.5 ±0.5 (5) 2.9 ±0.9 (4) 1.0 2.7 ±0.3 (10) 2.1 ±0.2 (10) 2.0 ±0.3 (9) 2.5 ±0.3 (10) 2.0 4.0 ±0.5 (10) 3.9 ±0.5 (10) 2.8 ±0.3 (10) 3.7 ±0.5 (10) 3.0 2.9 ±0.4 (5) 2.6 ±0.3 (5) 1.9 ±0.4 (5) 2.6 ±0.3 (5) 4.0 2.6 ±0.5 (5) 2.4 ±0.8 (4) 1.1 ±0.3 (4) 2.6 ±0.5 (5) Table 3. E f f e c t s o f i s o f l u r a n e on t h e a m p l i t u d e s of AHPs o f CA- p y r a m i d a l neurons. MIU, P. 32 AHP duration (s ± SEM, no. of a p p l i c a t i o n s ) IFL Perfusion time (min) (Vol. Control I s o f l u r a n e Recovery %) 3 6 6 0.5 2.7 ±0.5 (4) 1.9 ±0.6 (4) 2.1 ±0.7 (4) 2.8 ±0.9 (4) 1.0 4.8 ±0.6 (6) 3.8 ±0.7 (5) 2.9 ±0.5 (9) 3.9 ±0.7 (5) 2.0 4.7 ±1.0 (9) 4.3 ±1.3 (9) 3.5 ±1.1 (9) 4.3 ±1.0 (8) 3.0 3.5 ±0.5 (5) 2.2 ±0.6 (5) 1.2 ±0.5 (4) . 3.1 ±0.9 (5) 4.0 3.8 ±1.1 (4) 3.1 ±1.3 (3) 3.0 ±0.9 (4) 3.5 ±1.1 (4) Table 4. E f f e c t s o f i s o f l u r a n e on t h e d u r a t i o n s o f AHPs o f CA. p y r a m i d a l neurons. MIU, P. FIG. 8. E f f e c t s of i s o f l u r a n e on AHPs of 18 CA^ pyr a m i d a l neurons. (A) Time- and dose-dependent r e d u c t i o n of the amplitudes of AHPs by i s o f l u r a n e . AHP amplitudes, measured from 18 CA^ neurons, were expressed as the percentage of the maximum AHP amplitude obtained by e l i c i t i n g 4 or 5 s p i k e s . S i g n i f i c a n t r e d u c t i o n i n the amplitudes o f AHPs were observed a f t e r ~6 min p e r f u s i o n s of 2-4% i s o f l u r a n e . (B) Reduction of the d u r a t i o n s of AHPs by i s o f l u r a n e . AHP durations, measured from 18 CA^ neurons, were expressed as the percentage of the maximum AHP d u r a t i o n o b t a i n e d by e l i c i t i n g 4 or 5 s p i k e s . Means ± SEM; *, p<0.05 by Student t - t e s t . MIU, P. 34 I I C D Control K 3 3 min • • 6 min Recovery 0.5 1.0 2.0 3.0 4.0 Isoflurane concentration (Volume %) B V 0.5 1.0 2.0 3.0 4.0 Isoflurane concentrations (Volume %) MIU, P. 35 Nonetheless, profound reductions in maximum AHP duration were s t i l l observed at 6 min with application of 1-3 %isoflurane. These were not st a t i s t i c a l l y significant (Student t-test) because of the large variability in the meas-urements, which is partly attributable to the spontaneous synaptic activity and electrical 'noise'. 3.6 Synaptic transients Postsynaptic potentials were generated in 11 pyramidal CA^ cells by stimulating the afferent fibres in the stratum radiatum (Fig. 2) . In all of these c e l l s , subthreshold current strength was used to e l i c i t EPSPs of near maximal amplitude without generating action potentials. At least 20 of these EPSPs were averaged by computer to give means for the control, and for each time interval of isoflurane perfusion, including recovery. The aver-aged EPSP amplitude of the 11 cells in the control solution was 6.7 ± 0.3 mV (35 averages; cf. Table 5, representing actual values). As illustrated in Fig. 9A, the amplitudes of EPSPs were reduced substantially after 6 min perfusion of 3 % isoflurane; at this time, a slight hyperpolarization K 2 mV) was observed. The amplitudes of EPSPs recovered at 6 min after returning the perfusion to the control ACSF. More than one dose of isoflurane was applied to 11 c e l l s , and the results indicate that the amplitudes of the EPSPs were reduced significantly and in a dose- and time-dependent manner with increasing doses {0.5-3%) of isoflurane (Fig. 9B). Full recovery, measured after 6 min of returning to ACSF, was observed with low concentrations; however, only partial recovery was observed with high concentrations (Fig. 10). Inhibitory postsynaptic potentials (IPSPs), following EPSPs, were meas-ured using K-acetate electrode in 8 out of 11 cells that received 22 appli-cations of isoflurane in various concentrations. The data show that despite depolarization of the membrane potential, isoflurane at 0.5%reduced the MIU, P. 36 Evoked EPSP amplitude (mV ± SEM, no. of applications) IFL (Vol. %) Control 3 Perfusion time (min) Isoflurane 6 3 Recovery 6 0.5 6.8 ±0.7 (8) 6.0 ±0.8 (8) 5.1 ±0.9 (8) 5.7 ±1.0 (8) 6.4 ±1 . 1 (7) 1.0 6.4 ±0.6 (8) 5.3 ±0.5 (8) 4.6 ±0.5 (9) 5.6 ±0.8 (9) 6.8 ±0 .8 (9) 1.5 6.1 ±0.6 (6) 4.7 ±0.8 (6) 3.3 ±0.7 (6) 3.6 ±0.6 (6) 4.7 ±0 .6 (6) 2.0 5.8 ±0.5 (7) 3.9 ±0.3 (7) 2.8 ±0.4 (7) 2.9 ±0.2 (7) 4.3 ±0 .4 (6) 3.0 8.5 ±0.6 (5) 6.3 ±1.2 (5) 3.1 ±1.2 (4) 4.0 ±1.4 (4) 6.0 ±1 .5 (4) 4.0 9.8 (1) 6.6 (1) 2.2 (1) 2.7 (1) 6.1 (1) Table 5 . E f f e c t s of i s o f l u r a n e on the e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s of CA.. pyramidal neurons. MIU, P. FIG. 9. I s o f l u r a n e reduces the amplitudes of EPSPs i n a dose- and time-dependent manner. (A) C o n t r o l , 6 min of 3% i s o f l u r a n e , and recovery t r a c e s (-25 computer averaged traces) were superimposed. P e r f u s i o n of ~6 min of 3% i s o f l u r a n e d r a s t i c a l l y reduced the peak amplitude of EPSPs and a s s o c i a t e d c e l l h y p e r p o l a r i z a t i o n (2 mV). P a r t i a l recovery to c o n t r o l peak amplitude was observed at -6 min a f t e r r e t u r n i n g the p e r f u s i o n t o normal ACSF. Orthodromic s t i m u l a t i o n (10-15 V, 0.2 Hz) of the S c h a f f e r c o l l a t e r a l s was used. KAc e l e c t r o d e . (B) Dose-response curve o b t a i n e d from 11 CA^ neurons exposed t o v a r i o u s doses of i s o f l u r a n e . The peak amplitudes of the EPSPs were expressed as percentage of maximum peak EPSP amplitude. Open c i r c l e s represent 3 min of i s o f l u r a n e p e r f u s i o n , and c l o s e d c i r c l e s represent 6 min of i s o f l u r a n e p e r f u s i o n . Means ± SEM; a, s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l ; b, s i g n i f i c a n t l y d i f f e r e n t from 3 min of i s o f l u r a n e p e r f u s i o n ; p<0.05 (Post hoc m u l t i p l e comparison). O v e r a l l s i g n i f i c a n c e determined by ANOVA. MIU, P. 38 MIU, P. 39 FIG. 10. Time c o u r s e of i s o f l u r a n e a c t i o n and r e c o v e r y on evoked EPSPs. Dose-dependent p a r t i a l r e c o v e r y was o b s e r v e d i n CA^ neurons exposed t o h i g h a n a e s t h e t i c c o n c e n t r a t i o n s . Means ± SEM. MIU, P. P e r f u s i o n t i m e (min ) MIU, P. 41 peak amplitudes of IPSPs, whereas in concentrations of 1 % or more, the peak IPSP amplitude was enhanced (Fig. 11). The reduction in IPSP amplitude with 0.5 % administration indicates possible physiological anaesthetic action since depolarization increases CI - electrochemical gradients, thereby f a c i l i t a t i n g C l ~ influx. The time course of recovery from these effects was similar to that observed in the case of isoflurane-depression of EPSPs. MIU, P. 42 if FIG. 11. E f f e c t s of i s o f l u r a n e on i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l s (IPSPs). Responses of the amplitudes of IPSPs t o va r i o u s c o n c e n t r a t i o n s of i s o f l u r a n e were c a t e g o r i z e d by membrane p o t e n t i a l response f o l l o w i n g 6 min p e r f u s i o n . MIU, P. 43 CD B CO cu CO ^ ; CO CD O H CD c CO o 1 5 0 T 1 0 0 -5 0 -0 Isoflurane EZ3 0.5 % ESS 1.5 % CSD 2.0 % T - 5 0 X X X X X X X x. Hyperpolarization Depolarization MIU, P. 44 4. DISCUSSION 4.1 Isoflurane concentrations Knowledge of the concentration administered is important for comparing these results with the experimental findings reported by other investiga-tors, as well as to correlate them to clinical observations. In the present investigations, the concentrations of isoflurane applied to the hippocampal slices are in a range similar to those used c l i n i c a l l y . For clinical appli-cations, typical MAC values for anaesthesia range between 0.5% and 2.0% (Goodman and Gil man, 1985; Miller, 1986). These values correspond to the vaporizer outputs of 0.6-2.3% for isoflurane or 0.4-1.5% for halothane. Using chromatographic methods, some investigators have found that 0.2-1.0 mM isofurane in the liquid phase corresponded to 0.6-3.4 %isoflurane in the gas phase (Yoshimura et a l , 1985; Takenoshita and Takahashi, 1987). Noninvasive 1 Q fluorine-NMR techniques also have been used for in vivo studies to assess anaesthetic concentraions in the brain (Wyrwicz et al , 1983; Romp-painen et a l , 1986; Mills et a l , 1987; L i t t et a l , 1987). Accordingly, endotracheal applications of 1% isoflurane resulted in 0.1-0.5 mM in the rabbit brain (Wyrwicz et a l , 1983). The values of 0.02-0.3 mM isoflurane obtained in our experiments, with vaporizer outputs of 0.5-4%, can be attributed to loss of anaesthetics into the atmosphere above the recording chamber, and/or a result of other techni-cal d i f f i c u l t i e s associated with collecting the samples (e.g., incomplete sealing of NMR sampling tubes). However, the bath concentrations of iso-flurane applied to our hippocampal slices were probably sufficient to give final tissue concentrations similar to those found in the CSF of patients anaesthetized with isoflurane (Goodman and Gil man, 1985; Yoshimura et a l , 1985; Bazil et a l , 1987). For instance, Bazil et al (1987) reported that MIU, P. 45 the distribution of a chemically very similar anaesthetic agent, halothane, in animals anaesthetized at a 1% vaporizer setting is f a i r l y uniform - 32.3 nmol/mg l i p i d in whole brain, and 35.1 nmol/mg l i p i d in a slice preparation of the hippocampus. Since the l i p i d solubility is similar between isoflur-ane and halothane, the distribution of isoflurane in the hippocampal slices i s , therefore, assumed to be similar to that in whole brain. Hence, the effects of isoflurane on the membrane electrical properties of CAj pyrami-dal cells presently observed have cl i n i c a l 'relevance', i.e., some bearing on the actual mechanism of the general anaesthetic state. 4.2 Postsynaptic modifications 4.2.1 Resting potentials and membrane conductances. The present inves-tigations have demonstrated inconsistent changes in resting potentials of guinea pig CAj c e l l s . However, in agreement with previous reports, a hyperpolarization was consistently observed in the presence of TTX. Furthermore, isoflurane had no effect on the input conductances either in the absence or presence of TTX. The inconsistency in resting potentials observed in the absence of TTX was probably related to endogenous excitatory and/or inhibitory afferent ac t i v i t i e s . The slicing technique invariably alters the balance between excitatory and inhibitory influences impinging on a target c e l l . Conse-quently, each recording will reflect the net tonic influence predominating in each neuron following slicing. For example, in the presence of a strong tonic inhibitory influence, the resting potential is likely to be 'clamped' close to the reversal potential of IPSPs. Addition of TJX would be expected to abolish spontaneous presynaptic action potentials responsible for the tonic excitatory and/or inhibitory influences. In view of the above, the effects of isoflurane on resting membrane potential would be more accurately reflected in recordings with TTX present in the perfusion media. Under MIU, P. 46 these conditions, the impaled cell was, therefore, dissociated from synaptic bombardments caused by presynaptic action potentials. It was clear that isoflurane hyperpolarized cells without concomitant changes in their input conductances. The lack of isoflurane effect on membrane conductance, meas-ured in the soma, does not necessarily mean that anaesthetic actions do not occur on the membrane electrical properties of CA^ c e l l s , as reported by others. It is conceivable that isoflurane may alter membrane conductances in the dendritic area which presumably would not be detected due to the cable properties of the dendrites, i.e., opening of ionic channels thereby creating a distal shunt in the axonal membrane. Previous investigations have demonstrated that intravenous agents (e.g., pentobarbital) and volatile anesthetics in cl i n i c a l doses reduce cellular excitability by increasing membrane conductance and producing a hyperpolarization. These effects have been observed in the a-motoneurons (Whitney and Glenn, 1986; Takenoshita and Takahashi, 1987) and hippocampal pyramidal cells (Nicoll and Madison, 1982; Berg-Johnsen and Langmoen, 1987; O'Beirne et a l , 1987). In contrast, there are some reports of no change in the input conductances or the resting potentials of rat CA^  pyramidal cells (Yoshimura et a l , 1985; Brooks et a l , 1986). Nicoll and Madison (1982) postulated that the increased input conductance produced by anaesthe-tics like halothane, diethylether, and pentobarbital, may be mediated by potentiating a g K. In support with this proposal, volatile anaesthetics (Chalazonitis, 1967), pentobarbital (Sato et a l , 1967), and metabolic inhi-bitors (e.g., 2,4-dinitrophenol) (Godfraind et a l , 1970; Godfraind et a l , 1971) reduce neuronal excitability, presumably mediated by an increase in g K. Recently, the role of g K in the regulation of neuronal excitability during anoxia has been confirmed by the findings that reversal of the electro-chemical gradient for CI" had no effect on anoxia-induced events MIU, P. 47 (Hansen et a l , 1982), and that the anaesthetic evoked hyperpolarization was markedly enhanced in potassium-free media (Fujiwara et a l , 1987). 4.2.2 Afterhyperpolarizing potentials. In the present investigation, isoflurane reduced the amplitudes of the AHPs in a dose-dependent manner. This effect is consistent with other observations of isoflurane-actions in human sympathetic ganglia (Puil et a l , 1988) and neocortical cells (El-Beheiry and Puil, 1988) in which the amplitudes of AHPs also were suppressed. The AHPs are prolonged hyperpolarizing undershoots, rebounding after spike-like depolarizations. Associated with the hyperpolarizations are an increase in membrane conductances. The mechanism for this phenomenom was f i r s t determined in spinal motoneurons (Krnjevic and Lisiewicz, 1972; Krnjevic et a l , 1978; Zhang and Krnjevic, 1986), and later in various other neurons, e.g., sympathetic (Kuno et a l , 1983; Pennefather et al_, 1985; Tokimasa, 1985), inferior olivary (Llinas and Yarom, 1981), olfactory cortex (Constanti and Sim, 1987), and hippocampal pyramidal cells (Kandel and Spencer, 1961; Schwartzkroin, 1975; Alger and Nicoll, 1980; Hotson and Prince, 1980). In the hippocampal pyramidal c e l l s , these AHPs were attributed to the 2+ + . activation of a Ca -dependent K -conductance (g K) since like moto-neurons (Krnjevic et a l , 1978), they could be abolished by intracellular injection of EGTA (Schwartzkroin and Stafstrom, 1980), removal of external 2+ Ca (Brown and G r i f f i t h , 1983), or the addition of external divalent 2+ 2 + 2 + 2 + cations (e.g., Cd , Mn , and Co ) which interfered with Ca entry (Alger and Nicoll, 1980; Hotson and Prince, 1980; Wong and Prince, 1981; Brown and G r i f f i t h , 1983; Lancaster and Adams, 1986; Segal and Baker, 1986). The genesis of AHPs is strongly dependent on the extracellular [K +] levels (Alger and Nicoll, 1980). Hence, anaesthetics that interfere 2+ with Ca metabolism, for example, will alter the AHPs. MIU, P. 48 Our findings further support this possibility since AHPs were diminish-ed by isoflurane. Nevertheless, i t is feasible that isoflurane may act at 2+ other sites which are involved in the Ca -dependent g^, for example, + CI and K channels. Blockade of CI conductances by anaesthetics, however, is unlikely or at least may be masked since other investigations have demonstrated that anaesthetics potentiate IPSPs (Nicoll et a l , 1975; Gage and Robertson, 1985; Proctor et a l , 1986; Harrison et a l , 1987). El-Beheiry and Puil (1988) observed that, in the neocortical c e l l s , Cl~ conductances contribute slightly to the genesis of AHPs since addition of bicuculline only slightly reduced the amplitudes of AHPs. Thus far, we are not aware of any evidence showing that anaesthetics directly block K cur-rents in mammalian CNS. In support of this view is the observation that action potential durations were not altered during applications of isoflur-ane. Isoflurane-induced reductions in the amplitudes of AHPs occur at mem-brane potentials where there would be no contribution of membrane voltage. 2+ Such an influence might be expected from the observations that Ca - a c t i -vated K + channels show a voltage dependency. Applications of pentobarbital (O'Beirne et al_, 1987) and ethanol (Carlen et a l , 1982) increase the AHP amplitudes in hippocampal CA^ pyra-midal c e l l s . These findings, however, are not necessarily incompatible with our results using isoflurane. The inconsistency suggests that different agents may alter calcium metabolism in a different manner, for example: changes in the disposition of intracellular Ca (Krnjevic, 1974; Ho and 2+ Harris, 1981) or in Ca -currents (Blaustein and Ector, 1975; Blaustein, 1976; Heyer and Macdonald, 1982; Krnjevic and Puil, 1988). In the present investigation, isoflurane is not likely to have produced 2+ alterations in g^'s other than the Ca -dependent g K since input con-ductances were not reduced significantly. Furthermore, isoflurane applica-MIU, P. 49 tions had no effects on the amplitudes and durations of spontaneous and evoked spikes which suggests that Na+-channels are unlikely postsynaptic sites of anaesthetic action. The failure of isoflurane applications to affect input conductance may suggest possible involvement of other postsyn-aptic g^'s or alterations in presynaptic mechanisms. 4.3 Presynaptic modifications 4.3.1 Axonal conduction. The variability in resting potentials and, in particular, the reductions in the amplitudes of EPSPs observed in the pres-ent investigations suggest possible alterations in axonal conduction pro-duced by anaesthetics. Sowton and Sherrington (1905) showed that c l i n i c a l concentrations of volatile anaesthetics block synaptic transmission but have a relatively minor influence on axonal conduction. This observation has been subsequent-ly confirmed in other studies on using sympathetic nerve axons (Larrabee and Posternak, 1952), lateral olfactory tract fibres (Richards et a l , 1975), and perforant path of the hippocampus (Richards and white, 1975). Berg-Johnsen and Langmoen (1986) recently demonstrated that the amplitude and conduction velocity of action potentials in the unmyelinated fibres were reduced by isoflurane in a dose-dependent manner, within the stratum radiatum layer of the hippocampus. The apparent contradiction in the anaesthetic actions on axonal conduc-tion may be attributed to the high anaesthetic doses applied to the slice preparations in the investigations of Berg-Johnsen and Langmoen (1986) because isoflurane was delivered directly from the vaporizer output (see above and wyrwicz et a l , 1983; Bazil et a l , 1987). Moreover, Bazil (ly87) observed that for similar levels of anaesthesia in in vivo and in vitro preparations, anaesthetic concentrations in the equilibrating gas were lower than those used for the in vivo preparations. Hence, high concentrations of MIU, P. bU anaesthetic may have reduced both axon terminal excitability and conduction + velocity, presumably by enhancing inactivation of a Na -conductance (Saint et a l , 1986; Quastel and Saint, 1986). The bath concentrations of isoflur-ane achieved in our experiments were less than 0.3 mM which suggests that anaesthetic actions on the transmitter release mechanism are more likely, at least in the isoflurane dose range of 0.5 to 2%. 4.3.2 Synaptic transmission. The findings that isoflurane may affect neuronal transmission by interfering with synaptic potentials are generally consistent with those reported for other anaesthetics observed in mammalian spinal cord (Brooks and Eccles, 1947; Somjen and G i l l , 1963; Loyning et a l , 1969), sympathetic ganglion (Larrabee and Posternak, 1952), cuneate nucleus (Galindo, 1969), and olfactory cortex (Richards, 1973). Recent investiga-tions have shown that halothane induces a uniform suppression of both EPSPs and IPSPs in rat spinal motoneurons (Takenoshita and Takahashi, 1987). How-ever, Yoshimura et al (1985) found that c l i n i c a l doses of isoflurane, halo-thane, and enflurane had no effect on the amplitudes of EPSPs of hippocampal CA^  neurons, although these agents selectively suppressed the peak ampli-tudes of IPSPs. In the same type of preparation, injectable anaesthetics including barbiturates (Nicoll et a]_, 1975), etomidate (Proctor et a l , 1986), and alphaxalone (Harrison et a l , 1987) have been reported to poten-tiate IPSPs. Accordingly, and using voltage-clamp techniques, Gage and Robertson (1985) demonstrated that halothane applications reversibly increase the time constant of decay of hippocampal inhibitory postsynaptic currents. A reduction in IPSP amplitude was observed in the present inves-tigation but only with an isoflurane concentration of less than IX How-ever, and in concert with the above findings of Gage and Robertson and others, concentrations of isoflurane more than or equal to 1 % enhanced the amplitudes of IPSPs. MIU, P. 51 The dose-dependent suppression and augmentation of the peak IPSP ampli-tude by isoflurane suggest possible contamination of the amplitudes of the IPSPs by the ionic mechanism generating the EPSPs. That i s , high doses of the anaesthetic reduced the amplitude of EPSPs thereby unmasking the fu l l amplitudes of IPSPs. However, changes in the amplitudes of IPSPs may be measured more exactly during applications of isoflurane in low anaesthetic doses that do not significantly alter the amplitudes of EPSPs (Yoshimura et a l , 1985). Hence, we may speculate that the excitatory phase of anaesthesia observed c l i n i c a l l y may be a consequence of the suppression of IPSPs preced-ing that of EPSPs. 4.4 Mechanism of anaesthesia 4.4.1 Membrane effects. The dose-dependent effect of isoflurane on responses such as synaptic transients, AHPs, and membrane polarization sug-gest a possible role of concentration-dependent anaesthetic actions in the membrane. A good correlation between anaesthetic potency and l i p i d solubil-ity has led to the conclusion that the principal target site of the anaes-thetic agents is in the hydrophobic region of membrane bound protein or in the l i p i d bilayer (La Bella, 1981; Franks and Lieb, 1982). Moreover, anaes-thesia (or hypnosis) results when an equivalent molar concentration of a particular agents is achieved within the cell membrane (Meyer, 1937; Mull ins, 1954; Seeman, 1972) or when equivalent volumes of cell membrane are occupied by the anaesthetic molecules (Richard, 1978; Koblin, 1979). Accum-ulation of isoflurane in the membrane can be inferred from present observa-tion on the dose-dependent suppression of the peak amplitudes of synaptic transients and AHPs, e.g., the greater the dose, the greater the depression of the amplitudes of EPSPs. The time required for isoflurane to accumulate in the plasma membrane is another important determining factor for the concentration dependent drug MIU, P. 52 action. This was also demonstrated indirectly by the observations that the degree of peak amplitude reduction in EPSPs and AHPs was greater at 6 min than that at 3 min of isoflurane administration. An augmentation in the drug effect with prolonged perfusion time was, nevertheless, an expected phenomena because of the high lipo p h i l i c i t y of the agent and time-dependent accumulation of isoflurane in the plasma membrane. Thus from the synaptic transient studies, partial recovery of peak EPSP amplitude after high iso-flurane concentrations indicates an accumulation of an appreciable number of isoflurane molecules in the l i p i d bilayer. Consequently, a longer time would be required for the anaesthetic molecules to overcome the 'barrier' of l i p o p h i l i c i t y and exist the plasma membrane. In contrast, f u l l recovery after low concentrations of isoflurane indicates a rapid termination of anaesthetic action on membrane proteins or ionic channels. 4.4.2 Transmitter release. It is well established that depolarization of the presynaptic terminals leads to transmitter release via inward move-2+ ment of Ca through voltage-dependent calcium channels (Hodgkin and Keynes, 1957; Katz and Miledi, 1967a,b; Katz and Miledi, 1969; Augustine et a l , 1987). Most of these investigations were performed using presynaptic terminals of the squid giant synapse (Katz and Miledi, 1969; Katz and Miledi, 1967b; Llinas et a l , 1981; Augustine and Eckert, 1984; Augustine et a l , 1987), but relatively few studies have been performed in the mammalian CNS because of the inaccessibility of these terminals to direct electro-2+ physiological analysis using present techniques. Nevertheless, Ca -de-pendent synaptic transmission has been demonstrated in the guinea-pig olfactory cortex (Richards and Sercombe, 1970) and hippocampus (Dingledine and Somjen, 1981). MIU, P. 53 Using radioactive labelled Ca 2 + ( 4 5Ca), anaesthetic doses of barbi-45 turates have been shown to reduce Ca uptake into the presynaptic termin-als (synaptosomes) isolated from mammalian brain (Blaustein and Ector, 1975; Sohn and Ferrendelli, 1976; Ondrusek et a l , 1979) and sympathetic ganglia (Blaustein, 1976). Recently, Krnjevic and Puil (1988) demonstrated that 2+ halothane reduces Ca -inward currents in hippocampal CA^ pyramidal ce l l s . An accumulation of internal Ca , [Ca ]., inactivates the voltage-gated calcium conductance (Eckert and Ewald, 1982), resulting in 2+ reduced Ca influx. There is evidence demonstrating that volatile anaes-2+ thetics can, indeed, elevate intracellular free Ca by suppressing mi to-ot chondrial Ca -uptake (Rosenberg, 1973; Sweetman and Esmail, 1975; Krnjevic, 1975). Although Ca -currents were not directly measured in the present investigation as in the experiments of Krnjevic and Puil (1988), the reduction in the amplitudes and durations of AHPs suggests that presynaptic 2+ calcium metabolism is perhaps altered by isoflurane. A Ca -dependent-g^ has been described in synaptosomes of rat brain inferring its existence in terminal membrane (Bartschat and Blaustein, 1985). In view of these observations, anaesthetics may produce impairment of synaptic transmission 2+ by reducing presynaptic Ca entry (Blaustein and Ector, 1975; Blaustein, 1976; Ondrusek et a l , 1979; Heyer and Macdonald, 1982) and subsequent trans-mitter release (Haycock et a l , 1977). Hence, the attenuation of synaptic transmission during isoflurane anaesthesia is l i k e l y to be mediated, at 2+ least in part, by a suppression of EPSPs secondary to a reduction in Ca -dependent transmitter release. Rapid recovery from the actions of isoflurane in the hippocampal slices was observed within minutes; however, complete elimination of such agents in an in vivo preparation apparently requires a considerably longer time (Wyrwicz et a l , 1983; Li t t et a l , 1987). This is partly due to the large MIU, P. 54 amount of l i p i d in the brain tissue, acting as a 'reservoir' for r e - d i s t r i -bution of the anaesthetic molecules. Hence, the long period for recovery of cognitive functions in elderly patients from the effects of general anaes-thesia (cf. Introduction) may result from persistence of the anaesthetic in 2+ the CNS, thereby prolonging the disruption of Ca -dependent synaptic transmission in aged brain (cf. Landfield and P i t l e r , 1984). MIU, P. 55 5. SUMMARY AND CONCLUSIONS (i) The effects of isoflurane on synaptic potentials and membrane excita-bi1ities in hippocampal slice in vitro preparations of guinea pig were investigated in CA^  neurons using intracellular recording techniques. A relatively new c l i n i c a l anaesthetic, isoflurane, was applied in the a r t i f i c i a l cerebrospinal fluid bathing the in vitro preparations. 19 ( i i ) Isoflurane concentrations were detected in the bath with fluorine 19 nuclear magnetic resonance techniques ( F-NMR), and were found to range between 0.02 to 0.3 mM. These concentrations are similar to the doses of isoflurane used c l i n i c a l l y for producing the general anaes-thetic state in humans, ( i i i ) Stable recordings, lasting 1-5 hours, were obtained from 82 CA^  pyramidal c e l l s . In a random sampling of 23 of the 82 neurons, the mean resting membrane potential was -65.4 ± 1.3 mV, and input resis-tances usually were greater than 50 Mfi. Spontaneous activity was sometimes observed. In order to study the postsynaptic effects of isoflurane administration without interference from endogenous synaptic activity, tetrodotoxin (1 uM) was added to the perfusate. The combined application of tetrodotoxin and isoflurane usually hyper-pol ari zed the neurons by 3-5 mV. However, no significant changes in their input resistances were observed, (iv) The average number of applications of isoflurane to the neurons was 1.8. Doses of isoflurane in the range of 0.02-0.3 mM produced no consistent effects on resting potentials; however, the number of neurons that were depolarized by isoflurane administration was signi-ficantly less at high doses than at low doses. Similarly, input resistances which were determined using bridge-balance techniques from MIU, P. 56 the membrane voltage displacements resulting from intracellular injections of hyperpolarizing current pulses and from the slopes of current-voltage relations, were not significantly altered by (0.5-4%) administrations of isoflurane. (v) Applications of isoflurane reduced the amplitudes and durations of afterhyperpolarizing potentials following multiple spikes evoked by injections of current pulses. The amplitudes of excitatory and inhib-itory postsynaptic potentials generated by subthreshold electrical stimulation of the Schaffer collaterals also were diminished by such administrations. These effects on afterhyperpolarizing potentials and synaptic transients were s t a t i s t i c a l l y significant as well as dose-and time-dependent. (vi) It is concluded that isoflurane anaesthesia does not greatly affect the passive membrane properties of hippocampal CA^ neurons. 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